Current HIV-1 vaccine candidates based on recombinant adenovirus serotype 5 (rAd5) vectors are promising but may prove limited by the high prevalence of pre-existing anti-Ad5 immunity in the developing world. To overcome this problem, we have constructed a series of novel serotype and novel chimeric rAd vectors that effectively circumvent anti-Ad5 immunity. We have also demonstrated that heterologous rAd prime-boost regimens utilizing two serologically distinct rAd vectors elicit remarkably potent immune responses, particularly regimens involving rAd26 priming and rAd5 or rAd5HVR48 boosting. In addition, we have generated rAd Vectors expressing optimal C clade, M consensus, and M mosaic HIV-1 antigens designed to minimize genetic distance and to optimize T cell epitope coverage among global HIV-1 sequences. We propose to develop a practical, two-injection, heterologous rAd26 prime, rAd5HVR48 boost regimen expressing antigens optimized for global coverage as a novel candidate HIV-1 vaccine. The goals of Project 1 are to select the optimal antigens for our multivalent HIV-1 vaccine candidate and to evaluate the immunogenicity and protective efficacy of the rAd26 prime, rAd5HVR48 boost regimen in rhesus monkeys to support the clinical program in Project 2 and the manufacturing program in Project 3. In Project 1, we hypothesize that M consensus or M mosaic HIV-1 antigens will prove superior to naturally occurring C clade and VRC multiclade antigens for immunologic coverage of diverse global virus sequences. We further hypothesize that the optimal vaccine regimen expressing SIV antigens will afford significant protection against both homologous and heterologous SIV challenges in rhesus monkeys. To explore these hypotheses, we propose the following four Specific Aims: 1. To compare the magnitude and breadth of immune responses elicited by optimal C clade, M consensus, M mosaic, and VRC multiclade antigens in rhesus monkeys; 2. To determine the protective efficacy of the optimal vaccine regimen expressing various sets of SIV antigens against homologous SIV challenges in rhesus monkeys with anti-Ad5 immunity; 3. To evaluate the protective efficacy of the optimal vaccine regimen against heterologous SIV challenges in rhesus monkeys with anti-Ad5 immunity; and 4. To assess the mucosal immunogenicity and protective efficacy of the optimal vaccine regimen against multiple, low-dose, mucosal SIV challenges in rhesus monkeys with anti-Ad5 immunity.